The overall objective of this research program is to establish the role of human sperm enzymes and inhibitors in the fertilization process and to develop this knowledge into clinically applied methodology. During the coming project period, it is the first objective to carefully reexamine some commonly accepted properties of the human acrosin system, e.g.: if acrosin has a single or multiple catalytic sites; if proacrosin is a zymogen that requires proteolytic cleavage for activation or is an inactive form of an allosteric enzyme for which activation represents a substantial increase in catalytic activity due to molecular rearrangement and/or cofactors; and if the inhibition of acrosin in acrosomal extracts at neutral or basic pH is entirely due to the dialyzable acrosin inhibitor(s) known to be associated with spermatozoa or also to other factors. Some other controversial properties of acrosin and proacrosin will also be investigated. As the second objective, the synchronous acrosome reaction system developed for human spermatozoa during the current grant period will be validated further. A number of enzymes reputed to be involved in the acrosome reaction of nonhuman species, will be critically evaluated for their function in the human sperm acrosome reaction. Comparative studies will be performed with guinea pig spermatozoa. Specifically, the quantitative activity and role of the following enzymes will be investigated: 1) adenylate cyclase; 2) cyclic nucleotide phosphodiesterase; 3) acrosin/proacrosin; 4) phospholipase A2; 5) cyclooxygenase; 6) thromboxane synthase; 7) prostacyclin synthase; 8) PGH isomerase; and 9) lipoxygenase. As a third objective, the clinical applicability of previous findings will be assessed. These studies will include further evaluation of the biochemical and physiological mechanisms of action of aryl 4-guanidinobenzoates (synthesized acrosin inhibitors that have potential as contraceptives), the development and evaluation of improved diagnostic methodology to assess the fertilizing capacity of human semen (hypoosmotic swelling test, acrosin test and acrosome reaction test), and the evaluation and improvement of cryopreservation techniques for human sperm. The proposed research should lead to a better understanding of the mechanisms of human fertilization and to new and/or improved clinically useful technology.